Control of heart rate is a therapeutic goal with proven benefit for treatment of a wide range of conditions. Heart rate acceleration via surgically implanted electronic pacemakers is the only effective treatment for age-related sinoatrial node dysfunction (SND), whereas heart rate reduction via a general blocker of hyperpolarization- activated, cyclic nucleotide-sensitive (HCN) channels is emerging as an important treatment for angina and heart failure. While these treatments are effective, they also have important limitations. Hence, substantial effort has been invested towards development of biological pacemakers via expression of HCN channels and towards heart rate lowering drugs that block sinoatrial HCN channels. However, progress towards these goals is limited by a lack of detailed mechanistic information about the sinoatrial node-specific HCN4 channel isoform. HCN4 produces the funny current, if, in sinoatrial node myocytes (SAMs), mutations in HCN4 are associated with SND, and modulation of If in SAMs is already a proven means to control heart rate. Results from the previous funding period and new preliminary data establish the existence of two novel, isoform- specific activators of HCN4. The central goals of this proposal are 1) to define the molecular and biophysical mechanisms by which these novel regulators control HCN4 channels, and 2) to determine how these regulators impact, and can be used to control, pacemaker activity in SAMs. These goals will be pursued using an array of techniques that leverage the complementary expertise of the research team. Protein interactions and post-translational modification of HCN4 channels will be assayed using protein affinity chromatography, fluorescence resonance energy transfer, X-ray crystallography, and comparative proteomic profiling. Biophysical mechanisms underlying the new regulatory modes will be defined by patch clamp electrophysiology of heterologous-expressed wild type and mutant HCN4 channels. Reagents that mimic and inhibit the new regulators are being developed and will be assessed for their effects on If and pacemaker activity in patch clamp recordings from acutely-isolated mouse SAMs. If successful, these experiments will provide novel and detailed mechanistic information about isoform-specific regulation of HCN4 and will deliver proof-of-concept data for the ability of small molecules to alter pacemaker activity via these mechanisms. Results of these studies could guide development of new treatment paradigms for control of heart rate.